The possible participation of superoxide radicals in biochemical hydroxylation reactions will be investigated. Particular attention will be given to the mechanism of the side-chain hydroxylation of phenylethlyamine derivatives catalyzed by dopamine-beta-hydroxylase, the enzyme catalyzing the conversion of dopamine to norepinephrine. This study is significant in that it may yield important information regarding the mechanism by which dopamine-beta-hydrolase and other mono-oxygenases incorporate molecular oxygen in their substrates. The elucidation of this mechanism may help to clarify the etiology of Parkinson's disease, a neuro-enzymologic disorder in which there is a specific deficit of nigro-striatal dopamine. The identity of the active oxygen utilized by dopamine-beta-hydroxylase will be investigated by: (1) examination of the electron paramagnetic resonance spectra at low temperature of the reduced enzyme in the presence and absence of oxygen (with and without the use of spin traps), (2) determination of fluorescence produced by the addition of chemiluminescent scavengers of superoxide radical to the enzyme using fluorescence spectroscopy with photon counting capability, (3) a determination of the reactivities of the oxidized and reduced enzyme with the superoxide radical by means of pulse radiolysis and stopped-flow photolysis, and by (4) an examination of the effect of the addition of scavengers of superoxide radicals or singlet oxygen to the hydroxylation system. A comparative study of the reactions of the superoxide radicals or singlet oxygen to the hydroxylation system. A comparative study of the reactions of the superoxide radical with substrates and non-substrates of dopamine-beta-hydroxylase under a variety of conditions will be carried out with production of superoxide radical via 60Co gamma-irradiation or stopped-flow photolysis. Product analyses will utilize high pressure liquid chromatography and ultraviolet/visible absorption spectroscopy. We anticipate that these experiments will provide significant insight into the mechanism by which dopamine-beta-hydroxylase effects the conversion of dopamine of norepinephrine, and that this insight may help clarify the etiology of Parkinson's disease.